The Gram-positive soil bacterium Bacillus thuringiensis is well known for its production of proteins or delta-endotoxins, that are toxic to a variety of lepidopteran, coleopteran, and dipteran larvae. Different strains of B. thuringiensis have been shown to produce different insecticidal crystal proteins, which are specifically toxic to certain species of insects (reviewed by Höfte and Whiteley, 1989; Schnepf et al., 1998).
The specific toxicity of insecticidal toxins produced by B. thuringiensis for target insects and their non-toxicity to plants and other organisms has made compositions comprising different Bt strains the product of choice for the biological control of agricultural insect pests. Various of the genes encoding the crystal proteins have been cloned and their DNA sequences determined (Höfte and Whiteley, 1989; Crickmore et al., 1995). This has led to the engineering of modified delta-endotoxin encoding genes and the development of plants expressing these delta-endotoxin genes to make them insect resistant.
The family of cry1 genes encode the Cry1 crystal proteins, which are primarily active against lepidopteran pests. The protoxin form of Cry1 delta-endotoxins comprises a C-terminal protoxin part, which is not toxic and is thought to be important for crystal formation (Arvidson et al., 1989). The amino-half of the protoxin comprises the active toxin segment of the Cry1 protein. Different domains have further been identified in the active toxin, which are implied in different aspects of the toxicity effect (Grochulski et al., 1995). However, these functions seem to be dependent on the delta endotoxin examined.
Significant effort has gone into modifying the cry1 genes to improve expression levels in plants while at least retaining their toxicity to the target insects. Modification of the cry1Ab and cry1Ac genes to remove putative plant polyadenylation signals and instability motifs (without altering the encoded amino acid sequences) resulted in increased resistance of the plants transformed with these sequences (van der Salm et al., 1994). Modifications of the cry1Ab gene have been described in U.S. Pat. No. 6,320,100, U.S. Pat. No. 6,180,774 and U.S. Pat. No. 6,114,608. U.S. Pat. No. 5,500,365 describes how modification in the 240 region of the coding region of a cry1Ab gene so as to remove putative plant signals is of significant importance to increase expression levels and thereby toxicity of the Cry toxin in plants.
The present invention relates to a novel modified Cry1Ab protein and DNA sequences encoding this protein, which can be used to engineer insect resistance in plants. More particularly, it was found that this modified sequence, despite having a native 240 region, ensures sufficiently high expression in plant cells to confer insect resistance to the plant or plant tissue in which it is expressed.